The cystatin superfamily of cysteine proteinase inhibitors is comprised of three major families: Type 1 cystatins, which are cytosolic and include stefins A and B; Type 2 cystatins, which are present in most bodily fluids and include cystatins C, D, E, F, and S; and Type 3 cystatins, which are present in plasma and include the kininogens and fetuin (1, 2). Collectively, these molecules inactivate cysteine proteinases and thus regulate (i) bone resorption, neutrophil chemotaxis, and tissue inflammation, (ii) hormone processing and antigen presentation, and (ii) resistance to bacterial and viral infections (1-3). Cystatin C (CystC) is a ubiquitously expressed, small molecular weight (˜16 kDa) secretory protein that preferentially inactivates cathepsin B, a cysteine proteinase implicated in stimulating cancer cell invasion and metastasis (4, 5), and in activating latent TGF-β from inactive ECM depots (6, 7). Through its conserved cysteine protease inhibitor motif, CystC binds and inactivates cathepsin B by forming a reversible, high affinity enzyme-inhibitor complex (8, 9). Independent of its effects on cathepsin B activity, CystC also regulates cell proliferation (10, 11), raising the possibility that CystC targets proteinase-dependent and -independent pathways.
Mutations in or altered expression of CystC has been linked to the development and progression of several human pathologies. For instance, a single point mutation in CystC causes Hereditary CystC Amyloid Angiopathy, a lethal autosomal dominant disease that results in massive cerebral hemorrhages in early adulthood (12). Moreover, altered CystC expression or serum enzyme-inhibitor levels are used as diagnostic markers for chronic renal insufficiencies (13), and for cancers of the lung, skin, colon, and myeloid compartment (3, 14-16). Thus, altered CystC concentrations within cell microenvironments have dire consequences leading to the development and progression of human diseases.
TGF-β is a multifunctional cytokine that governs cell growth and motility in part through its regulation of cell microenvironments, and thus plays a prominent role in regulating disease development in humans (17). Critical to regulation of cell microenvironments by TGF-β is its induction or repression of cytokines, growth factors, and ECM proteins by fibroblasts (17).
Transforming growth factor-β (TGF-β) is also a potent suppressor of mammary epithelial cell (MEC) proliferation, and as such, an inhibitor of mammary tumor formation. However, aberrant genetic and epigenetic events operant during tumorigenesis typically abrogate the cytostatic function of TGF-β, thereby contributing to tumor formation and progression. For example, malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, thus enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients.
Oncogenic epithelial-mesenchymal transitions (EMT) comprise a complex array of gene expression and repression that elicits tumor metastasis in localized carcinomas (Thiery, 2002; Grunert et al., 2003). The acquisition of metastatic phenotypes by dedifferentiated tumors is the most lethal facet of cancer and the leading cause of cancer-related death (Yoshida et al., 2000; Fidler, 2002). Transforming growth factor-β (TGF-β) normally represses these processes by prohibiting epithelial cell proliferation, and by creating a cell microenvironment that inhibits epithelial cell motility, invasion, and metastasis (Blobe et al., 2000; Siegel, 2003). Carcinogenesis often subverts the tumor suppressing function of TGF-β, thereby endowing TGF-β with oncogenic activities that promote the growth and spread of developing tumors, including the initiation and stabilization of tumor EMT (Thiery, 2002; Grunert et al., 2003; Blobe et al., 2000; Siegel et al., 2003; Wakefield et al., 2002).
The duality of TGF-β to both suppress and promote cancer development was observed originally using transgenic TGF-β1 expression in mouse keratinocytes, which initially suppressed benign skin tumor formation prior to promoting malignant conversion and spindle cell carcinoma generation (Cui et al., 1996). More recently, TGF-β signaling was shown to inhibit the tumorigenicity of normal, premalignant, and malignant breast epithelial cells, while stimulating that of highly invasive and metastatic breast cancer cells (Tang et al., 2003). Fundamental gaps exist in the knowledge of how malignant cells overcome the cytostatic actions of TGF-β, and of how TGF-β stimulates the progression of developing tumors. Indeed, these knowledge gaps have prevented science and medicine from developing treatments effective in antagonizing TGF-β oncogenicity in progressing cancers, particularly those of the breast.
TGF-β is widely expressed during development to regulate the interactions between epithelial and mesenchymal cells, particularly those in the lung, kidney, and mammary gland. Inappropriate reactivation of EMT during tumorigenesis is now recognized as an important process necessary for tumor acquisition of invasive and metastatic phenotypes (Thiery, 2002; Grunert et al., 2003). By cooperating with oncogenes and growth factors, TGF-β potently induces EMT and serves to stabilize this transition via autocrine signaling. Moreover, these events appear to underlie TGF-β oncogenicity and its ability to promote cancer progression (Miettinen et al., 1994; Oft et al., 1998; Oft et al., 1996; Portella et al., 1998). Molecular dissection of TGF-β signaling systems necessary for its induction of EMT has clearly established a role for Smad2/3 in mediating EMT, particularly when coupled with signals emanating from oncogenic Ras (Piek et al., 1999; Oft et al., 2002; Janda et al., 2002). However, Smad2/3-independent signaling also has been implicated in TGF-β stimulation of EMT. For instance, TGF-β stimulates EMT in cancers of the breast and other tissues by activating PI-3-kinase, AKT, RhoA, p160 (ROCK), and p38 MAPK (Janda et al., 2002; Bhowmick et al., 2001, Mol. Biol. Cell; Bhowmick et al., 2001, J. Biol. Chem.; Bakin et al., 2000; Yu et al., 2002) In addition, EMT in TGF-β treated MECs is abrogated by measures that inhibit β1 integrin activity (Bhowmick et al., 2001, J. Biol. Chem.), thus establishing the necessity of β1 integrin expression for EMT stimulated by TGF-β. Finally, by repressing Id2 and Id3 expression (Kowanetz et al., 2004), inducing Snail and SIP1 expression (Kang et al., 2004), and stimulating NF-κB activity (Huber et al., 2004), TGF-β regulates transcription factor activity operant in mediating the transition from epithelial to mesenchymal cell markers. Clearly, EMT and the mechanisms whereby TGF-β participates in this process involve a complex cascade of gene expression and repression, the magnitude of which remains to be elucidated fully.
Accordingly, although TGF-β clearly inhibits the growth and development of early stage tumors, an accumulating body of evidence implicates TGF-β signaling as a stimulus necessary for the metastasis and dissemination of late stage tumors (Blobe et al., 2000; Siegel, 2003). A comprehensive understanding of how TGF-β both suppresses and promotes tumorigenesis remains an unknown and fundamental question that directly impacts our ability to effectively target the TGF-β signaling system during treatment of human malignancies. Indeed, deciphering this paradox remains the most important question concerning the biological and pathological actions of this multifunctional cytokine. The ability of TGF-β to induce cancer growth and metastasis suggests that developing therapeutics to antagonize and/or circumvent TGF-β signaling may prove effective in treating metastatic malignancies, perhaps by preventing TGF-β stimulation of EMT.